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Urgent: Please help with dilution factor???

  • 16-12-2012 6:50pm
    #1
    winnie2012
    Registered Users, Registered Users 2 Posts: 7 ✭


    Im trying to figure out a procedure but Im having trouble with the serial dilution factor. Sorry this is a bit long winded.

    Method 1:
    20 ul of compound x (1000ng/20ul or 50,000ng/ml) is added to milk (1g). 4 mls of methanol is added. After shaken and centrifugation the methanol layer is removed and evaporated under nitrogen. The extract is resuspended in 1 ml of ethyl acetate. 100 ul of this is diluted in 900 ul of buffer before analysis.
    This results in a final concentration 50 ng/ml in the extract.

    Method 2:
    20 ul of compound x (1000ng/20ul or 50,000ng/ml) is added to milk (1g). 4 mls of methanol is added. After shaken an centrifugation the methanol layer is removed and evaporated under nitrogen. The extract is resuspended in 1 ml of hexane and 250 ul of water/solvent mix. After shaken and centrifugation 200 ul of water/solvent mix is removed and evaporated to dryness. The extract is resuspended in 1 ml of ethyl acetate. 100 ul of this is diluted in 900 ul of buffer before analysis.
    This resulted in a final concentration of 40 ng/ml in the extract.

    I can’t understand the dilution factor. It looks like compound x was diluted by 1000 in method 1 and 1250 in method 2 but how?


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